The present invention relates to improved nucleic acid processing devices of all types, and more particularly to miniaturized DNA processing devices with surface treatments designed to reduce DNA adsorption to device surfaces exposed to DNA containing mediums.
A recent development in the fields of analytical chemistry and biotechnologyhas been the miniaturization of devices and systems for the processing and analysis of DNA. See e.g., McCormick, et al., 1997. Anal. Chem. 69:2626. Similarly, the current trend towards microfabrication has been driven by efforts to parallel the miniaturization accomplished in the semiconductor industry, and to exploit similar microfabrication techniques. See e.g., Ramsey, et al., 1995. Nature Med. 1:1093. Justifications for miniaturization include reduced cost, increased speed and reliability, distributed access (point-of-care diagnostics), decreased sample and reagent consumption and reduced waste generation.
An example of a microfabricated DNA analysis device is set forth in PCT Publication WO 96/35810, which is hereby incorporated by reference in its entirely. This aforementioned publication describes electrophoresis devices for the separation and observation of biopolymer fragments in an electrophoretic gel. In one embodiment, an electrophores is device is disclosed which possesses miniaturized electrophores is lanes, which are formed by open channels in a flat plate having dimensions down to approximately 25 xcexcm and closed by a flat cover plate. In a further embodiment, the publication discloses an electrophores is device which includes an integrally-associated, miniaturized reactor for generating biopolymer fragments for subsequent separation by the device. These miniaturized features are capable of being constructed by various micro-machining techniques, including the lithographic and etching methodologies initially developed in the semiconductor industry.
A further example of a microfabricated DNA analysis device is set forth in the commonly-assigned, U.S. patent application Ser. No. 08/623,346, filed Mar. 27, 1996, which is hereby incorporated by reference in its entirety. This aforementioned application discloses an apparatus for the separation of charged particles in a medium according to the differential diffusion properties of the particles within the electrophoretic medium by use of a spatially- and temporally-varying electric potential. Such an apparatus has application to the separation of single-stranded or double-stranded DNA fragments. In one embodiment, the device consists of a series of miniaturized electrodes which are patterned on a substrate and a cover plate which has one or more miniaturized channels (also down to approximately 25 xcexcm). This device is also described as being fabricated using the techniques initially developed within the semiconductor industry.
Due to their decreased dimensions the ratio of surface to volume in miniaturized or microfabricated DNA processing devices is markedly increased over other conventional devices. See e.g., Shoffner, et al., 1996. Nuc. Acids Res. 24:375. This increased surface-to-volume ratio increases the significance of effects of surface chemistry in such microfabricated devices. In particular, it is well known in the art that DNA interacts strongly with and adheres to a number of surfaces. See e.g., Hjerten 1985. J. Chromatography 347:191. The hydrophilic phosphate groups and hydrophobic protonated bases mean that almost any surface is likely to interact. In addition, the harsh processes used during the standard microfabrication process can damage or contaminate the surfaces creating even stronger interaction forces. See e.g., Henck, 1997. Tribology Letters 3:239. Although this problem is present in larger scale DNA processing devices, it is considerably exacerbated in micro-machined devices with larger surface to volume ratios. It is also a problem in DNA processing systems such as PCR reactors, capillary and plate gel electrophoresis systems.
Surface interactions have been addressed for a microfabricated polymerase chain reaction (xe2x80x9cPCRxe2x80x9d) device (see e.g., Shoffner, et al., 1996. Nuc. Acids Res. 24:375. Additionally, several types of surface treatments were investigated in an initial attempt to find PCR xe2x80x9cfriendlyxe2x80x9d surfaces, including surface treatment by silanization followed by a polymer treatment, by stoichiometric silicon nitride coating, and by silicon oxide coating. It should be noted, however, that only silicon oxide was demonstrated not to inhibit the PCR reaction; whereas the inhibition of the PCR amplification reaction by the other treatments methodologies was presumed to have been the result of surface binding sites that non-specifically adsorbed molecules involved in the PCR reaction (see e.g., Cheng, 1996. Nuc. Acids Res. 24:380.
Accordingly, it is apparent that there is a need for surface treatments for surfaces created in micro-machined DNA processing devices that inhibit DNA surface adsorption. Such an inhibition is termed herein surface xe2x80x9cpassivation.xe2x80x9d
It should be noted that citation of references herein is not to be taken as an admission that such references are prior art to the instant invention.
The present invention discloses the finding that certain surface treatments possess the ability to reduce DNA adsorption. For example, certain surface treatments are based upon: (i) plasma-enhanced, low temperature deposition of silicon oxide or (ii) low pressure chemical vapor deposition (hereinafter designated xe2x80x9cLPCVDxe2x80x9d) of low temperature silicon oxide. In particular, the present invention discloses conditions (including precursors), deposition process conditions and subsequent process conditions, which provide for minimal adherence of DNA to a treated surface. Similarly, other surface treatments of the present invention are based upon LPCVD deposition of silicon nitride. In particular, the present invention discloses the finding that certain levels of silicon-enrichment possess novel and highly efficacious properties. Furthermore, the present invention discloses the finding that surface treatments, based upon low pH wash solutions, also markedly reduce DNA adsorption. These aforementioned treatments, as discloses herein, have been adapted to microfabrication processes which have, heretofore, only been utilized in the fabrication of miniaturized devices, primarily in the electronics industry.
Accordingly, one embodiment of the present invention discloses methodologies for the administration of these surface treatments to various types of DNA analysis devices. Another embodiment of the present invention discloses devices made by these methodologies which may be applied, for example, to the analysis of nucleic acids. In a preferred embodiment, the devices are improved DNA processing devices possessing surfaces to which have been administered the surface treatments and washes of the present invention. As these treatments reduce DNA adsorption, such improved devices may be advantageously further miniaturized with an attendant increase in the overall surface to volume ratios.
In a further embodiment, the present invention includes methods for assaying the extent of DNA adsorption to untreated and treated surfaces. These methods include, but are not limited to, washing the surfaces with fluorescently-labeled DNA, rinsing, and fluorescence detection of adsorbed DNA by use of a spectrofluorometry.
The instant invention also may be applied to both microfabricated and to larger scale DNA systems and devices. Such systems and devices may perform processing functions including, but not limited to: (i) DNA analysis (e.g., sequencing, separation, hybridization, electrophoresis; (ii) DNA processing (e.g., DNA replication, polymerase chain reaction (xe2x80x9cPCRxe2x80x9d), Reverse Transcription-basedPCR (RT-PCR), ligase chain reaction (xe2x80x9cLCRxe2x80x9d), in vitro transcription and translation, strand exchange with or without enzymes); (iii) DNA modifications (e.g., end- or internal-labeling,phosphorylation, de-phosphorylation,digestion, ligation, multiplex formation for strand identification); (iv) DNA packaging (e.g., linking to form higher ordered structures); and (v) DNA extraction.
In one embodiment, the present invention discloses a method for quantitatively ascertaining the level of adsorption of a nucleic acid to a surface comprising the steps: (i) contacting the treated surface with a solution of labeled nucleic acid molecules; (ii) washing this contacted surface so as to remove the labeled nucleic acid solution and (iii) measuring the amount of label still present on the treated surface. In a preferred embodiment, the nucleic acid is DNA and the label is fluorescent. The aforementioned treated surface is comprised of a nucleic acid processing device which contacts a medium containing nucleic acids, wherein the solution comprises said medium containing nucleic acid molecules in a concentration expected to be present in said device, and wherein the medium is allowed to contact the surface for a time representative of times that the medium contacts the surface during operation of the device.
In a second embodiment, the present invention discloses an apparatus for processing of nucleic acids which is comprised of one or more surfaces contacting a nucleic acid-containing medium and a surface film upon which is deposited a silicon-rich, silicon nitride deposit. Preferably, the silicone nitride surface film is deposited by a method comprising chemical vapor deposition. In one aspect of this embodiment, the silicon-rich, silicon nitride surface has the chemical composition, SiNx, where that X is selected to minimize nucleic acid adsorption to the surface(s). In a preferred embodiment, X is a valve between approximately 0.8 and approximately 1.2, or, more preferably, is a valve between approximately 0.95 and approximately 1.05. In another aspect of this embodiment, the silicon-rich, silicon nitride possesses an index of refraction between approximately 2.1 and approximately 2.5, and more preferably between approximately 2.15 and approximately 2.25.
In a third embodiment, the present invention discloses an apparatus for the processing of nucleic acids comprising: one or more surfaces which contact a nucleic acid-containing medium and a surface film coating which is comprised of silicon oxide (possessing 1-4% by weight of hydroxyl groups and less than 0.5% by weight of hydride groups) deposited on the surface(s) by a chemical vapor deposition methodology.
In a fourth embodiment, the instant invention includes a method for producing an apparatus for processing nucleic acids comprising the step of depositing a coating of silicon oxide on one or more surfaces of said device that contact a medium containing nucleic acids, wherein the deposition is by chemical vapor deposition methodology performed at a temperature selected so as to minimize nucleic acid adsorption to the surface(s). This deposition temperature is preferably less than 500xc2x0 C., more preferably less than 200xc2x0 C., or most preferably less than 100xc2x0 C.
In a fifth embodiment, the present invention discloses an apparatus for the processing of nucleic acids generated according to the methodology set forth in the fourth embodiment.
In a sixth embodiment, the present invention discloses a method for producing a device for processing nucleic acids comprising the step of washing the surface(s) of the device which contact a nucleic acid-containing medium with a specific washing solution. Preferably, the specific washing solution possesses a basic pH of at least 8, or is volatile, or is comprised of an oxidizing agent. In one aspect of the sixth embodiment, the specific washing solution comprises an alkalinizing agent selected from the group consisting of ammonium hydroxide (NH4OH) and sodium hydroxide (NaOH). In another aspect of the sixth embodiment, the specific washing solution comprises an aqueous solution of ammonium hydroxide (NH4OH) and the oxidizing agent hydrogen peroxide (H2O2), and preferably comprises a solution of approximately 4 parts of water, approximately 1 part of 30% NH4OH, and approximately 1 part of 30% H2O2. Alternately, the concentration of the NH4OH, the concentration of the H2O2, and the duration of the washing step are selected so as to minimize nucleic acid adsorption to the device""s surfaces. In a preferred embodiment, the step of washing occurs at room temperature.
In a seventh embodiment, the present invention discloses an apparatus for processing nucleic acids generated according to the methodology of the sixth embodiment.